Acrolein, a, highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed, reacts rapidly with and depletes cellular glutathione (GSH) content and is toxic to various types of cells. The long-term objective of this proposal is to advance our understanding of the molecular mechanisms of cell cycle control. The overall goal is to determine the mechanism by which sub-lethal doses of acrolein reduce proliferation in A549 cells. The hypothesis to be studied is that acrolein effects changes in the expression of one more growth-or stress- related genes or transcription factors secondary to a reduction in GSH. A large number of genes have been identified as having effects on cell division. Those selected for analysis in the current proposal were chosen based on preliminary results and published data suggestive for a role. The specific aims are: 1) to assess changes in the activation of the transcription factors AP-1 and NFkappaB and in the mRNA (northern blots) and protein levels (western blots) of gadd153, hsp70, c-jun, and c-fos at various times following acrolein insult and correlate these changes with decreases in GSH and proliferation; and 2) to determine if the decrease in A549 cell proliferation and the changes in gene expression or transcription factor activity following acrolein insult are a direct result of decreased cellular GSH and/or changes in the cellular redox state. Changes following acrolein treatment will be compared with changes seen following comparable GSH-depletion with alternative treatments (diethyl maleate, diamide and H202). Transcription factor activation will be studied using electrophoretic mobility gel shift assays. A recent report suggests that acrolein inhibits the activation of NFkappaB. Preliminary data confirm this report but do not yet reveal the mechanism. Studies examining IkappaB, p65 and p50 subunits, and nuclear translocation will address this issue.